1. Field of the Invention
The present invention relates to a method and apparatus for time-of-flight (TOF) mass spectrometry.
2. Description of Related Art
(a) Time-of-Flight Mass Spectrometer (TOF-MS)
A TOF-MS finds the mass-to-charge ratio (m/z) of sample ions by measuring the time taken for the ions to travel a given distance, based on the principle that the sample ions accelerated with a constant acceleration voltage have a flight velocity corresponding to the m/z. The principle of operation of the TOF-MS is illustrated in FIG. 26. The illustrated spectrometer has a pulsed ion source 5 composed of an ion generation portion 6 and a pulsed voltage generator 7.
Ions i present within the electric field are accelerated by the acceleration voltage generator 7. The accelerating voltage is a pulsed voltage. Acceleration caused by the acceleration voltage and time measurement performed by an ion detector system (including detector 9) are synchronized. Simultaneously with the acceleration caused by the accelerating voltage generator 7, the ion detector 9 starts to count the time. When the ions reach the ion detector 9, the detector 9 measures the flight time of the ions i. Generally, the flight time increases with increasing m/z. Ions having small values of m/z reach the detector 9 earlier and thus have shorter flight times.
The mass resolution of the TOF-MS is given by
                              mass          ⁢                                          ⁢          resolution                =                  T                      2            ⁢            Δ            ⁢                                                  ⁢            T                                              (        1        )            where T is the total flight time and ΔT is the peak width. That is, there are two major factors resulting in the peak width ΔT in the spectrum. One factor is the time focusing (ΔTf). The other factor is the response (ΔTd) of the detector. Assuming that both factors show a Gaussian distribution, Eq. (1) is given byMass resolution=T/2√{square root over ((ΔTf2+ΔTd2))}  (2)
If the peak width ΔT is made constant and the total flight time T can be elongated, the mass resolution can be improved. In practice, the response of the detector 9 is approximately 1 to 2 nsec. Therefore, the peak width ΔT is not reduced further.
A linear TOF-MS is very simple in structure. However, the total flight time T is on the order of tens of microseconds. That is, a very long total flight time cannot be achieved. Consequently, the mass resolution is not so high. One advantage of the linear type is that fragment ions produced during flight are almost identical in velocity with ions not yet fragmented (precursor ions). This makes it possible to read information only about the precursor ions from the mass spectrum.
FIG. 27 is a diagram illustrating the principle of operation of the reflectron TOF-MS. Identical components are indicated by identical symbols in both FIGS. 26 and 27. In the reflectron TOF-MS, an intermediate focal point is placed between the pulsed ion source 5 and a reflectron electric field 8. Time focusing is once done. Then, energy focusing is realized by the reflectron electric field 8 and the remaining free space. Thus, the total flight time can be prolonged to about 50 μsec without increasing the spectral peak width ΔT.
A point to be noticed in reflectron TOF-MS is the behavior of ions fragmented during flight. Since fragment ions are substantially identical in velocity with precursor ions, the kinetic energy of fragment ions is given by
  Up  ×      Mf    Mp  where Mf is the mass of the fragment ions, Mp is the mass of the precursor ions, and Up is the kinetic energy of the precursor ions. Therefore, depending on the mass Mf, kinetic energy differences much larger than the distribution of the initial kinetic energies of ions are produced. Since fragment ions are smaller in kinetic energy than precursor ions, the fragment ions make a turn earlier than the precursor ions within the reflectron field and reach the detector 9. This complicates the mass spectrum.
b) Multi-Turn TOF-MS
In the prior-art linear and reflectron types of TOF-MS, increasing the total flight time T, i.e., increasing the total flight distance, immediately leads to an increase in the size of the apparatus. An apparatus that has been developed to avoid increase in size of the apparatus and to realize high mass resolution is the multi-turn TOF-MS. The multi-turn TOF-MS is composed of plural electric sector fields, and ions are made to make multiple revolutions.
Multi-turn TOF-MS instruments are roughly classified into multi-turn TOF-MS in which ions repeatedly follow the same trajectory and helical trajectory TOF-MS in which the ion beam is made to describe a helical trajectory by shifting the trajectory plane every revolution. The total flight time T can be increased to milliseconds to hundreds of milliseconds, which may differ according to the flight distance per revolution and on the number of revolutions. High mass resolution can be accomplished with improved space saving design compared with the conventional linear and reflectron types of TOF-MS.
The multi-turn type is characterized in that ions are made to turn multiple times on a closed circulation trajectory. FIG. 28 illustrates the principle of operation of the multi-turn TOF-MS. In this apparatus, ions ejected from a pulsed ion source 10 are made to make many revolutions on an 8-shaped circuit trajectory formed by 4 toroidal electric fields. After the multiple turns, the ions are detected by a detector 15 (see, for example, non-patent reference 1). In this apparatus, 4 toroidal electric fields 12 are used. Each toroidal electric field is produced by combining a Matsuda plate with a cylindrical electric field. Thus, the 8-shaped circuit trajectory is created. Ions are made to turn multiple times on the trajectory, thus increasing the total flight time T.
Furthermore, this apparatus adopts an ion optical system that can fully satisfy the spatial focusing conditions and time focusing conditions whenever a revolution is made without depending on the initial position, initial angle, or initial energy (see, for example, patent reference 1). Therefore, the flight time can be prolonged without increasing time and spatial aberrations by causing ions to make multiple turns. The multi-turn type can realize space saving and high mass resolution but there is the problem that ions with small masses (having high velocities) surpass ions with large masses (having small velocities) because ions are made to repeatedly follow the same trajectory. This creates the disadvantage that the mass range is narrowed.
The helical trajectory TOF-MS is characterized in that the trajectory is shifted in a direction perpendicular to the circulation trajectory plane whenever one revolution is made, thus realizing a helical trajectory. In one feature of this helical trajectory TOF mass spectrometer, the starting and end points of the closed trajectory are shifted perpendicularly to the trajectory plane. To realize this, some methods are available. In one method, ions are introduced obliquely from the beginning (see, for example, patent reference 3). In another method, the starting and end points of the closed trajectory are shifted in the vertical direction using a deflector (see, for example, in patent reference 3). When viewed from a certain direction, the helical trajectory TOF-MS is the same as the multi-turn TOF-MS. Whenever one revolution is made, ions are made to descend, i.e., moved downward. As a whole, a helical trajectory is accomplished. This apparatus can solve the problem with the multi-turn TOF-MS (i.e., overtaking). However, the number of turns is restricted physically. Consequently, the mass resolution has an upper limit.
Fragment ions produced by fragmentation during flight cannot reach the detector, because electric sector fields act as kinetic energy filters. Therefore, a mass spectrum completely unaffected by fragment ions can be derived.
(c) MALDI (Matrix Assisted Laser Desorption/Ionization) and Delayed Extraction Technique
The MALDI is a method of vaporizing or ionizing a sample by mixing the sample into a matrix (such as liquid or crystalline compound or metal powder) having an absorption band at the wavelength of the used laser light, dissolving the sample, solidifying it, and illuminating the solidified mixture with laser light. In an ionization process which uses a laser and is typified by the MALDI, the initial energy distribution is wide when ions are created. To time focus the distribution, delayed extraction technique is used in most cases. This method consists of applying a pulsed voltage with a delay of tens of nanoseconds from laser irradiation.
FIG. 29A conceptually illustrates a general MALDI ion source and delayed extraction technique. The MALDI is a method of vaporizing or ionizing a sample 30 by mixing the sample into a matrix having an absorption band at the wavelength of the used laser light, dissolving the sample, solidifying it, and illuminating the solidified mixture with laser light. The sample 30 is adhered to the sample plate 20. A lens 1 (also indicated by numeral 23) receives the laser light. The light from the lens 1 is reflected by a mirror 24. The light reflected by the mirror 24 is made to hit the sample 30. As a result, the sample 30 is excited, producing ions. The ions are accelerated by accelerating electrodes 21 and 22 and introduced into a mass analyzer region.
A mirror 25, a lens 2, and a CCD camera 27 are disposed to permit observation of the state of the sample 30.
The sample 30 is mixed and dissolved in the matrix. The matrix is solidified. The solidified matrix is placed on the sample plate 20. Laser light is directed at the sample 30 through the lens 1 and mirror 24, vaporizing or ionizing the sample 30. The generated ions are accelerated by the accelerating electrodes 1 and 2 (21 and 22) and introduced into a TOF-MS. An electric potential gradient having a tilt as shown in (a) is applied between the accelerating electrodes 2 and 1 (22 and 21). After a delay of hundreds of nanoseconds, the potential gradient assumes the form as shown in FIG. 29B.
FIG. 30 is a diagram illustrating a time sequence using the prior-art delayed extraction technique. (a) indicates a laser beam. (b) indicates the electric potential at the accelerating electrode 1. (c) indicates measurement of the flight time. First, the accelerating electrode 1 and sample plate 20 are made equipotential. Then, the laser oscillates at instant t0. At instant t1, i.e., with a delay of hundreds of nanoseconds after receiving a notice signal from the laser indicating the oscillation, the voltage at the accelerating electrode 1 is varied from Vs to V1 at high speed. A potential gradient is created between the sample plate 20 and the accelerating electrode 1 to accelerate the ions. The potential at the accelerating electrode 1 returns from V1 to Vs at instant t2. Measurement of the flight time is started at instant t1 that is on the leading edge of the pulsed voltage. The measurement of the flight time ends at instant t3.
(d) Orthogonal Acceleration
In MALDI, ions are generated in a pulsed manner and so MALDI has very good compatibility with TOF-MS. However, there are numerous mass spectrometry ionization methods that produce ions continuously such as EI, CI, ESI, and APCI. Orthogonal acceleration has been developed to combine such an ionization method and TOF-MS.
FIG. 31 is a conceptual diagram of TOF-MS using orthogonal acceleration. This mass spectrometry is abbreviated oa-TOF-MS or oa-TOFMS. An ion beam produced from an ion source 31 that generates ions continuously is continuously transported into an orthogonal acceleration portion 33 with kinetic energy of tens of eV. In the orthogonal acceleration portion 33, a pulse generator 32 applies a pulsed voltage of tens of kV to accelerate the ions in a direction orthogonal to the direction of transportation from the ion source 31. The ions entering a reflectron field 34 are reflected by the reflectron field 34. In this way, the arrival time from the instant at which the pulsed voltage is started to be applied to the instant at which the ions arrive at the detector 35 is made different among different masses of ions. Consequently, mass separation is performed.
(e) MS/MS Measurement and TOF/TOF Equipment
In general mass spectrometry, ions generated from an ion source are mass separated by a mass spectrometer to obtain a mass spectrum. Information obtained at this time is only m/z values. This measurement is herein referred to as MS measurement in contrast to MS/MS measurement. In the MS/MS measurement, certain ions (precursor ions) generated from an ion source spontaneously fragment or are forcedly fragmented. The resulting product ions are observed.
In this measurement, information about the mass of the precursor ions and information about the masses of product ions produced along plural paths are obtained. Consequently, the information about the structure of the precursor ions can be obtained. FIG. 32 is a diagram illustrating MS/MS measurement. Precursor ions break into product ions 11, 12, 13, and so on. The structural analysis of the precursor ions is enabled by mass analyzing all the product ions.
A system consisting of two TOF-MS units connected in tandem is generally known as TOF/TOF equipment or TOF/TOF system and principally used in equipment making use of a MALDI ion source. The TOF/TOF equipment is composed of a linear TOF-MS and a reflectron TOF-MS. FIG. 33 conceptually illustrates MS/MS equipment in which the TOF-MS units are connected in tandem. In this example, the equipment consists of a linear TOF-MS 40 (first TOF-MS unit) and a reflectron TOF-MS 45 (second TOF-MS unit).
Ions exiting from an ion source 41 within the first TOF-MS unit pass through an ion gate 42 for selecting precursor ions. The time focal point of the first TOF-MS unit is placed near the ion gate 42. The precursor ions enter a collision cell 43, where they are fragmented. Then, the fragment ions enter the second TOF-MS unit. The kinetic energies of the product ions produced by the fragmentation are distributed in proportion to the masses of the product ions and given by
                    Up        =                  Ui          ×                      m            M                                              (        3        )            where Up is the kinetic energy of the product ions, Ui is the kinetic energy of the precursor ions, m is the mass of the product ions, and M is the mass of the precursor ions. In the second TOF-MS unit including a reflectron field, the flight time is different according to mass and kinetic energy. Therefore, product ions can be detected by a detector 46 and mass analyzed.
As one feature of the multi-turn TOF-MS is that an optical system is known which can fully satisfy the spatial and time focusing conditions without depending on the initial position, initial angle, or initial energy (see, for example, Patent reference 1). [Non-patent reference 1] Journal of the Mass Spectrometry Society of Japan, Vol. 51, No. 2 (No. 218), 2003, pp. 349-353 [Patent reference 1] Japanese patent laid-open No. H11-195398 (pages 3-4, FIG. 1) [Patent reference 2] Japanese patent laid-open No. 2000-243345 (pages 2-3, FIG. 1) [Patent reference 3] Japanese patent laid-open No. 2003-86129 (pages 2-3, FIG. 1).